P-TEFb promotes cell survival upon p53 activation by suppressing intrinsic apoptosis pathway

Abstract Positive transcription elongation factor b (P-TEFb) is the crucial player in RNA polymerase II (Pol II) pause release that has emerged as a promising target in cancer. Because single-agent therapy may fail to deliver durable clinical response, targeting of P-TEFb shall benefit when deployed as a combination therapy. We screened a comprehensive oncology library and identified clinically relevant antimetabolites and Mouse double minute 2 homolog (MDM2) inhibitors as top compounds eliciting p53-dependent death of colorectal cancer cells in synergy with selective inhibitors of P-TEFb. While the targeting of P-TEFb augments apoptosis by anti-metabolite 5-fluorouracil, it switches the fate of cancer cells by the non-genotoxic MDM2 inhibitor Nutlin-3a from cell-cycle arrest to apoptosis. Mechanistically, the fate switching is enabled by the induction of p53-dependent pro-apoptotic genes and repression of P-TEFb-dependent pro-survival genes of the PI3K-AKT signaling cascade, which stimulates caspase 9 and intrinsic apoptosis pathway in BAX/BAK-dependent manner. Finally, combination treatments trigger apoptosis of cancer cell spheroids. Together, co-targeting of P-TEFb and suppressors of intrinsic apoptosis could become a viable strategy to eliminate cancer cells.


Supplementary
. Selective inhibition of CDK7 and P-TEFb but not other tCDKs is syntheticlethal with MDM2 inhibitor Nutlin-3a.
(A) Compound classes of the FO5A oncology library. Pie chart represents classes of investigational and clinical anti-cancer compounds listed on the right.
(B) Dose-response viability curves of HCT116 cells treated with increasing doses of NVP-2 (green) alone and in combination with Nutlin-3a (10 M; light gold) or 5-Fluorouracil (5-FU; 25 M, dark gold) as indicated. Viability values obtained at 72 hr of the treatments using CellTiter-Glo 2.0 Cell Viability Assay were normalized to the values of DMSO-treated cells and are presented as percentages of the maximum viability which was set at 100 %. Results are presented as the average ± s.d. (n = 2).
(C) Waterfall plot representing results of the screen. Compounds with dDSS values ≥ 5 and ≤ -5 were considered as significant and depicted according to the legend on the right.
(D) (Top) 8 × 6 cytotoxicity matrix with combinatorial titrations of NVP-2 (green) with Nutlin-3a (red) at indicated doses in non-transformed CCD 841 CoN colon epithelial cells. Cytotoxicity values obtained at 48 hr of the treatments using CellTox Green Cytotoxicity Assay were normalized to the values of DMSO-treated cells and are presented as percentages of the maximum cytotoxicity which was set at 100 %. Results represent the average of independent experiments (n = 3). (Bottom) CCD 841 CoN cells were treated with the indicated combinations of NVP-2 (10 nM) and Nutlin-3a (10 M) for 8 hr prior to preparation of whole cell extracts and detection of the indicated proteins by Western blotting.
(E, H-K) 8 × 6 matrices with combinatorial titrations of iCDK9 (green) and the indicated tCDK inhibitors (black) with Nutlin-3a (red) at indicated doses to test for the synthetic lethality of compounds in HCT116 cells, depicting cytotoxicity (top) and synergy (bottom) of the combinations. Targeted tCDKs are indicated on top. Cytotoxicity values obtained at 48 hr of the treatments using CellTox Green Cytotoxicity Assay were normalized to the values of DMSO-treated cells and are presented as percentages of the maximum cytotoxicity which was set at 100 %. Results represent the average of independent experiments (n = 3).
(F,G) 8 × 6 matrices with combinatorial titrations of NVP-2 (green) with Nutlin-3a (red) and 5-Fluorouracil (5-FU; blue) at indicated doses to test for the synthetic lethality of compounds in HCT116 cells, depicting viability (top) and synergy (bottom) of the combinations. Viability values obtained at 48 hr of the treatments using CellTiter-Glo 2.0 Cell Viability Assay were normalized to the values of DMSOtreated cells and are presented as percentages of the maximum viability which was set at 100 %. Results represent the average of independent experiments (n = 3). Combinations with the highest Bliss synergy scores in HCT116 cells are highlighted (gold). Figure S2. Synthetic lethality of Nutlin-3a and antimetabolites with P-TEFb inhibitor NVP-2 is p53-dependent.

Supplementary
(A-D) 8 × 6 matrices with combinatorial titrations of NVP-2 (green) with Nutlin-3a (red) and 5-Fluorouracil (5-FU; blue) at indicated doses to test for the synthetic lethality of compounds in HCT116 and HCT116 TP53 -/cells, depicting viability (top) and synergy (bottom) of the combinations. Viability values obtained at 48 hr of the treatments using CellTiter-Glo 2.0 Cell Viability Assay were normalized to the values of DMSO-treated cells and are presented as percentages of the maximum viability which was set at 100 %. Results represent the average of independent experiments (n = 3). Combinations with the highest Bliss synergy scores in HCT116 cells are highlighted (gold). Figure S3. Activation of apoptosis underlies p53-dependent synthetic lethality of p53 activation and P-TEFb inhibition.

Supplementary
(A,B) HCT116, HCT116 TP53 R248W/+ and HCT116 TP53 R248W/cells were treated with DMSO and indicated combinations and doses of CDK9 inhibitors (green), Nutlin-3a (red), and 5-Fluorouracil (5-FU; blue) for 24 hr prior to preparation of whole cell extracts and detection of the indicated proteins by Western blotting. Figure S4. p53-induced genes of the intrinsic apoptosis pathway remain expressed under sub-lethal inhibition of P-TEFb.

Supplementary
(A) Scatterplots comparing raw read counts of all annotated genes from RNA-seq data sets (n = 3) are shown and spearman correlation (r) is indicated. HCT116 cells were treated for 8 hr as indicated on top of each graph.
(B) Gene set enrichment analysis of the protein-coding gene data set regulated by Nutlin-3a. The top gene set is shown. NES, normalized enrichment score. FDR, false discovery rate.
Supplementary Figure S5. Synthetic lethality of non-genotoxic p53 activation and P-TEFb inhibition depends on intrinsic apoptosis pathway and on-going Pol II transcription.
(A) 4 × 6 cytotoxicity matrices with combinatorial titrations of Nutlin-3a (red) with NVP-2 (green) at indicated doses of HCT116 cells co-treated with DMSO or Triptolide (1 M) as indicated. Cytotoxicity values obtained at 48 hr of the treatments using CellTox Green Cytotoxicity Assay were normalized to the values of DMSO-treated cells and are presented as percentages of the maximum cytotoxicity which was set at 100 %.
(B) Activity of Caspase 9 measured using Caspase-Glo 9 Assay in whole cell extracts of HCT116 cells treated with DMSO (grey), NVP-2 (3 nM; green) and Nutlin-3a (10 M; red) alone and in combination (gold), and with Triptolide (1 M; black) as indicated for 18 hr. Results are presented as luminescence values relative to the values of DMSO-treated cells and plotted as the mean ± s.e.m. (n = 4). **, P < 0.01, determined by Student's t test.
(C,D) HCT116 cells were treated with DMSO and increasing doses of NVP-2 and iCDK9 (green) for 24 hr prior to preparation of whole cell extracts and detection of the indicated proteins by Western blotting. Figure S6. Repression of genes encoding key components of the pro-survival PI3K-AKT pathway is a driver of the synthetic lethality of p53 activation and P-TEFb inhibition.

Supplementary
(A) HCT116 cells were treated with DMSO (grey) and Nutlin-3a (10 M; red) for 24 hr prior to quantifying mRNA levels of the indicated genes with RT-qPCR. Results normalized to the levels of GAPDH mRNA and DMSO-treated cells are presented as the mean ± s.e.m. (n = 3). *, P < 0.05, determined by Student's t test.
(C) HCT116 cells were treated with DMSO (grey) and NVP-2 (20 nM; green) for 3 hr prior to determining the levels of total Pol II at transcription start site (TSS) and gene body (GB) of the indicated genes with ChIP-qPCR. Results normalized to the values of IgG in DMSO-treated cells are presented as the mean ± s.e.m. (n = 2). *, P < 0.05; n.s., non-significant, determined by Student's t test.
Supplementary Figure S7. Combination treatments within the framework of p53 activation and P-TEFb inhibition trigger apoptosis of HCT116 spheroid cultures.

Source Identifier
Mouse monoclonal anti-CDK9 Santa Cruz Biotechnology